It is generally accepted that the phosphorylation- dephosphorylation of the 20,000-dalton light chains of myosin is an important component of the regulatory mechanism in smooth muscle. Initiation of contraction and an increase in the actin- activated ATPase activity of myosin require phosphorylation of the 20,000 dalton light chains of myosin by a myosin light chain kinase (MLCK). The reverse process, dephosphorylation, accoompanies relaxation and the loss of actin-associated ATPase activity. Although this general pattern for the function of myosin phosphorylation is well documented, this process is poorly understood at the molecular level. The experiments described in this proposal will characterize in detail the molecular mechanism of the regulation of smooth muscle contraction. First, we will elucidate how the phosphorylation of myosin regulates the ATPase activity of actomyosin (contraction- relaxation). Particularly the relationship between the phosphorylation of myosin, conformation of myosin and ATPase activity of actomyosin will be studied by several approaches. Second, the role of myosin light chains (20,000 dalton and 17,00 dalton) for the regulation of ATPase activity will be studied. Third, the physiological role of the phosphorylation of myosin by MLCK at a second site will be elucidated. We have shown that gizzard smooth muscle myosin is phosphorylated by MLCK not only at serine 19 of 20,000 dalton light chain but also threonine 18 of the light chain. We will further study this subject to clarify the physiological relevance of such an occurrance. Fourth, we will investigate the possible involvement of protein kinase C on the regulation of contractile system. Fifty, the phosphorylation-de-phosphorylation of actomyosin will be studied in different condition of actomyosin (attached state, actomyosin in fiber etc) to find possible regulation mediated by the substrate conformation. Sixth, we will study the structure-function relationship of MLCK. Seventh, the role of caldesmon for the regulation of smooth muscle contraction especially for the tension maintenance without phosphorylation of myosin will be studied. These experiments should permit a more detailed assessment of the role of myosin phosphorylation and to indicate the possible operation of other regulation mechanisms. It is anticipated that the overall understanding of regulation smooth muscle contractions will be significantly improved by the proposed studies.